Genetic deletions at the FHIT locus are the most frequent and the earliest known genetic change in lung cancer and several other common human cancers. The Fhit protein cleaves ApppA, a dinucleotide substrate, into AMP plus ADP, but we have shown that the ability of the enzyme to cleave ApppA does not correlate with tumor suppressor function. Rather, biochemical and genetic evidence indicate that Fhit signals for tumor suppression as an enzyme-substrate complex. To define the structural basis of signaling by Fhit protein, we co-crystallized a nonhydrolyzable ApppA analog with wild-type Fhit and with a mutant Fhit protein that has a defect in ApppA hydrolysis. As these crystals were hexagonal needles with a ~270 cell length, data collection at a synchrotron source was required. The crystal structures of Fhit-substrate analog complexes revealed a novel mode of protein regulation which bears similarities with proteins regulated by nucleotide-binding and with proteins regulated by protein phosphorylation. As has been seen for GTPases, the structure of the substrate complex is quite different from the product complex. However, unlike the case of GTPases, the protein portion of the complex is largely unchanged. Rather, Fhit presents two ApppA analogs on the "top" surface of the protein in place of a deep, positively charged groove. Fhit-ApppA complexes are, in fact, "phosphorylated" by six surface phosphates. Thus, crystal structure analysis suggests that Fhit-substrate complexes are the active, signaling form of Fhit. 4. C. Brenner, H.C. Pace, P.N. Garrison, A.K. Robinson, A. R sler, X. Liu, G.M. Blackburn, C.M. Croce, K. Huebner & L.D. Barnes, "Purification and Crystallization of Complexes Modeling the Active State of the Fragile Histidine Triad Protein," Protein Engineering, v. 10, pp. 1461-1463 (1997). H.C. Pace, P.N. Garrison, A.K. Robinson, L.D. Barnes, A. Draganescu, A. R sler, G.M. Blackburn, Z. Siprashvili, C.M. Croce, K. Huebner & C. Brenner, "Genetic, Biochemical and Crystallographic Definition of a Substrate Analog Complex with the Fragile Histidine Triad Protein as the Active Signaling Form of Fhit," Proc. Natl. Acad. Sci., v. 95, pp. 5484-5489 (1998).